Polarographic cell systems have met with wide acclaim particularly in the medical field, providing for detection and concentration measurement of many desired analytes. Enzymes are commonly used in such systems, especially in those situations wherein the analyte itself is not polarographically active but where a reaction product formed or reactant consumed by an enzymatic reaction with the analyte is polarographically active.
For example, in medical applications, one common procedure is to measure glucose in the blood of a patient. Typically, blood samples are withdrawn from the patient for an analysis for glucose concentration using a glucose oxidase electrode with a polarographic detector for detecting H.sub.2 O.sub.2 generated in accordance with the reaction: ##STR1##
The hydrogen peroxide generated by the reaction is measurable by a polarographic detector and, by appropriate calibration and calculation, glucose content in the sample can be accurately determined by the H.sub.2 O.sub.2 formed in the reaction.
The polarographic cell systems commonly used for these measurements include an enzyme containing laminated membrane that separates the analyte sample from the working electrode of the cell. These types of membranes are disclosed in the U.S. Pat. Nos. 3,979,274 and 4,073,713 (Newman), both patents being hereby incorporated by reference herein. In such membranes, a thin innermost membrane referred to as a barrier layer composed of cellulose acetate, silicone rubber, or methyl methacrylate is located adjacent the working electrode of the polarographic cell. Glucose oxidase enzyme is interposed between this barrier layer and an outer polycarbonate support layer. The outer support layer is typically about 5 um in thickness and is in contact with the analyte containing sample.
In a glucose analytical determination, glucose and oxygen permeate through the outer support layer and react in the presence of the enzyme. Hydrogen peroxide produced permeates through the inner barrier layer where it is polarographically detected. The support layer permits passage of glucose, oxygen and other molecules therethrough while not permitting passage of high molecular weight substances such as proteins, red blood cells and other macromolecules.
The barrier layer permits access of hydrogen peroxide to the working electrode while blocking passage of substances having molecular weights on the order of about 150 and greater such as ascorbic acid and uric acid.
It has been ascertained that in order to provide accurate linear measurement in solutions containing high glucose concentrations, such as in whole blood, plasma or serum, it is desirable to inhibit diffusion of the glucose to the enzyme layer relative to oxygen migration thereto. Otherwise, the ratio of glucose to oxygen contacting the enzyme is unfavorable and makes the oxygen content, rather than the glucose concentration of the sample, the rate limiting component of the reaction. In turn, this leads to inaccurate glucose concentration measurements by the instrument. Linearity, in such situations, occurs only over a range of low glucose concentration. This problem is not only limited to glucose determination but is also experienced in the measurement of other analytes, such as lactate.
In order to overcome this problem, it has been common practice to dilute the glucose and lactate concentrations of the sample so that the as measured analyte concentration level is within the range of concentration exhibiting linearity. However, it is often time consuming and impractical to dilute the analyte containing sample. Additionally, it is becoming commonplace to measure for analytes such as glucose or lactate on a single analytical testing device which incorporates other measurement channels as well. These other channels require undiluted whole blood or plasma as the analyte sample input; therefore requiring that the glucose and/or lactate measurement channel also function to measure analyte in the same undiluted whole blood or plasma sample medium.
In order to provide accurate glucose or lactate measurement in whole blood, the sample contacting membrane layer of the Newman type membranes has been modified to limit diffusion of the analyte to the enzyme layer. For example Japanese patent application Sho 59-40182 disclosed that the pore size of the outer sample contacting membrane should be 200 .ANG. or less, with tested 150 .ANG. pore sizes showing improved glucose measurement in whole blood samples. Later, in an obvious variation from the teachings of the Japanese reference, Young et al. in U.S. Pat. No. 4,759,828 indicated that the outer sample contacting layer should have pore sizes on the order of about 10-125 Angstrom units in diameter. The '828 patent expressly indicates that outer layers of 150 Angstrom unit pore size will not "sufficiently limit the diffusion of glucose molecules to allow glucose measurements to be made on undiluted serum."
In addition to the emphasis placed on small pore sizes in the outer, solution contacting layers of the laminated membrane, Vadgama et al. have emphasized the importance of low porosity materials. Percentage porosity is defined as the product of pore density.times.pore area.times.100. Porosities in the range of 0.001% or 0.005% to 0.5% and in general less than 2% are taught in Vadgama et al. E.P. Application 0 216 577. See also U.S. Pat. No. 5,437,973 (Vagdama et al.).
The move towards use of small pore size support layers has not been without problem. For example, during the fabrication of small pore size films of the type used as outer, solution contacting, layers in laminated enzyme containing membranes, the pores are often formed by an irradiation process in which fission fragments from an uranium atom or other forms of irradiation pierce the solid film precursor to form the desired film pore density. Pore sizes are established in a subsequent etching step using a strong alkali solution. In the final stages of this process, films are washed or treated with high molecular weight surfactants such as polyvinylpyrrolidone.
Poor uniformity has beet experienced when these films are incorporated into laminated enzyme containing membranes. It is thought that the large, bulky surfactant molecules used during the film fabrication processes tend to block or clog some of the pores. Because this can occur to different degrees within the relatively small unit area of the film which ultimately is used in a enzyme containing laminated membrane, relatively large membrane to membrane inconsistencies may be experienced. Small pore sizes, in general, also make it difficult to attain membrane uniformity during membrane manufacture.
Additionally, when small pore sized support membranes are used, the potential for clogging or obstruction during use in the analytical process is also present.
Accordingly, there is a need in the art for the provision of an enzyme containing laminated membrane assembly that is capable of measuring glucose and/or lactate in undiluted whole blood, plasma or serum which will not suffer from the aforementioned problems.